Thoracic aortic aneurysm occurs in approximately 50?70% of patients with bicuspid aortic valve (BAV), which affects 1?2% of the general population. There is a critical need to develop evidence to determine criteria for surgical intervention and to develop treatment that will prevent or reverse aneurysms for BAV patients. Thoracic aortic aneurysm in BAV frequently involves the proximal aorta, with smooth muscle cells (SMCs) that originate from neural crest stem cells (NCSCs). However, it spares the descending thoracic aorta, which has SMCs that originate from the paraxial mesoderm. The long-term goal of our research is to determine the molecular mechanisms responsible for the aortopathy associated with BAV and develop therapeutic strategies for aortic aneurysm in BAV. The objective of this research is to model BAV aortopathy in vitro using SMCs derived from BAV induced pluripotent stem cells (iPSCs) and in vivo using tissue-engineered vessels generated from these SMCs. Models will be used to explore treatments for aortic aneurysm in BAV. Our central hypothesis is that BAV aortic SMCs are defective in TGF-?-dependent differentiation of SMCs from NCSCs but not paraxial mesoderm and this defect causes decreased contractile function and secretion of extracellular matrix from these cells, resulting in aortopathy and subsequent aneurysm at the proximal aorta in BAV. Aim 1: We will characterize defects in vascular SMC differentiation in BAV/aneurysm cases in vitro. We will differentiate BAV and control iPSCs into NCSCs and paraxial mesoderm, then SMCs. We will determine the differentiation, contractility, and extracellular matrix of the BAV- and control-SMCs. We will define the key signaling pathway leading to defective differentiation in BAV NCSC-SMCs with a focus on canonical TGF-? signaling and myocardin transcription as identified in our pilot study. We will also perform RNA-seq to unbiasedly identify other potential pathways causing the defective differentiation of SMCs from NCSCs. We will rescue the defective differentiation of BAV SMCs from the NCSC lineage through pathways independent of the TGF-? pathway, such as rapamycin, which promotes SMC differentiation by inhibiting mammalian target of rapamycin and activating Akt2. Aim 2: We will determine the mechanism of aneurysm formation in vivo using nude rabbits that host engineered aorta generated from BAV patients? iPSCs. We will create tissue-engineered vessels populated with BAV NCSC-SMCs to replace the rabbits? abdominal aorta. We will determine the biomechanics and aneurysm formation of the engineered vessels in rabbits, and we will define differentiation and maturation of the SMCs and the TGF-? signaling of the engineered vessel. Finally, we will incorporate rapamycin into the scaffold to prevent the aneurysm in engineered vessels. Our human iPSC in vitro and in vivo model is innovative for studying the mechanisms of thoracic aortic aneurysm in BAV and for screening therapeutic agents. These studies will produce novel data on aortopathy, clarify mechanisms of aortic aneurysm formation in BAV patients, guide surgical intervention, and provide a foundation for future medical treatment of aortic aneurysm in BAV.